Fig 1.

Human NK cell line model of LYST deficiency mirrors CHS cell phenotype. A-G, Cellular phenotype of LYST-deficient NK cells. Fig 1, A, Transmission electron microscopy (TEM) images. G, Golgi apparatus; L, lysosomes; M, mitochondria; N, nucleus. Scale bars = 1 μm. Fig 1, B and C, Immunotransmission electron microscopy (Immuno-EM) of LAMP1 or perforin, respectively. Scale bars = 800 nm. Fig 1, D, Frequency distribution of perforin-positive granule sizes. Inset shows the average perforin-positive granule diameter. Fig 1, E, Average number of perforin granules. Fig 1, F, NK cells stained with antibodies against perforin (green) and different vesicular compartments (red): LAMP1 (late endosome/lysosomes), LAMP2 (lysosomes), CI-MPR (post–trans-Golgi network transport vesicles), or EEA-1 (early endosomes). Insets show differential interference contrast (DIC) images. Scale bars = 5 μm. Graphs show the percentage of perforin colocalization with vesicular markers. Fig 1, G, Frequency distribution of CI-MPR–positive or LAMP2-positive vesicle sizes. Insets show the average size of vesicles. H, Cytotoxicity of LYST-deficient NK cells at different effector/target (E:T) ratios. LYST levels were analyzed by using immunoblotting, and actin served as a loading control. I and J, Delivery of granzyme B from NK cells. The increase in granzyme B substrate fluorescence in target cells was monitored by using flow cytometry (see Fig E2, A). Fig 1, I, Percentage of target cells positive for granzyme B activity. Fig 1, J, Mean intensity of granzyme B substrate fluorescence in target cells. Data are shown as means + SDs. *P < .05, **P < .01, ***P < .001, and ****P < .0001, unpaired t test (Fig 1, D, E, and G) or 1-way ANOVA (Fig 1, I and J). Fig 1, D and E, n = 50 to 90 cells from 4 experiments; Fig 1, F, n = 18 cells from 2 experiments; Fig 1, G, n = 25 to 30 cells from 2 experiments; Fig 1, H-J, data from 4 experiments.